Volume 8, Issue 7, Pages 775-788 (July 2000) How a protein generates a catalytic radical from coenzyme B12: X-ray structure of a diol-dehydratase–adeninylpentylcobalamin complex  Jun Masuda, Naoki Shibata, Yukio Morimoto, Tetsuo Toraya, Noritake Yasuoka  Structure  Volume 8, Issue 7, Pages 775-788 (July 2000) DOI: 10.1016/S0969-2126(00)00164-7

Figure 1 Adenosylcobalamin, modes of cobalamin binding, and the coenzyme analogues used in this study. (a) Three-dimensional structure of adenosylcobalamin (coenzyme B12). (b) Base-off mode of cobalamin binding (methylmalonyl-CoA mutase) [2]. (c) Base-on mode of cobalamin binding (diol dehydratase) [5]. (d) cyanocobalamin and (e) adeninylpentylcobalamin. The parallelograms in (d) and (e) represent a corrin ring of cobalamins. Structure 2000 8, 775-788DOI: (10.1016/S0969-2126(00)00164-7)

Figure 2 Structure of the diol-dehydratase–adeninylpentylcobalamin complex. (a) Stereo drawing of the diol-dehydratase–adeninylpentylcobalamin complex showing the αβγ heterotrimer. Subunit α, red; β, green; γ, blue. Adeninylpentylcobalamin is located in the interface between the α and β subunits. (b) Surface representation of the adeninylpentylcobalamin-binding region. Subunit α, red; β, green. Figure 2b was drawn using MOLMOL [56]. Structure 2000 8, 775-788DOI: (10.1016/S0969-2126(00)00164-7)

Figure 3 Structure of the active site of the enzyme. (a) Stereo drawing of the active-site structure viewed from the direction perpendicular to the plane of the corrin ring of adeninylpentylcobalamin. (b) Stereo drawing of the active-site structure viewed from the direction perpendicular to that of (a). The eight β strands comprising the TIM barrel are numbered 1–8 from the N-terminal side. Adeninylpentylcobalamin is shown in the lower part of (b). Carbon atoms of adeninylpentylcobalamin and the substrate are colored in pale violet-red and green-yellow, respectively, and K+ is in cyan. This figure was generated using the program MOLSCRIPT [57] and the rendering program RASTER3D [58]. Structure 2000 8, 775-788DOI: (10.1016/S0969-2126(00)00164-7)

Figure 4 Hydrogen-bonding network in the adenine-ring-binding site and comparison of conformations of active-site residues around cobalamin. (a) Stereoview ball-and-stick model showing adeninylpentylcobalamin and the vicinity of the adenine ring. K+ is shown in cyan and the water molecules are in green. Hydrogen-bonding is shown as green dotted lines, and interactions between K+ and its ligands as dotted yellow lines. (b) Hydrogen-bonding scheme between adeninylpentyl group and the protein. The distances are indicated in Å. (c) Superimposition of the active site in the adeninylpentylcobalamin complex (blue) on that in the cyanocobalamin complex (red). The label ‘B’ after the residue number refers to residues of the β subunit. Figures 4a and c were generated using the program MOLSCRIPT [57] and the rendering program RASTER3D [58]. Structure 2000 8, 775-788DOI: (10.1016/S0969-2126(00)00164-7)

Figure 5 Stereo pairs of part of electron-density maps contoured for residues around the cobalamin and position-dependent changes in B factors before and after photoirradiation. (a) The map of diol-dehydratase–cyanocobalamin complex at 100K. The blue contours are drawn for the 2mFo–DFc map and the green contours, for the Fo–Fc map. (b) The map of diol-dehydratase–adeninylpentylcobalamin complex in the dark (1.7 Å resolution). (c) The map of diol-dehydratase–adeninylpentylcobalamin complex under the illuminated condition (1.75 Å resolution). (d) Comparison of B factors of atoms in the adeninylpentyl group. The red line shows values under the illuminated conditions, and the blue line shows values in the dark. The atom names on the horizontal axis are the same as those in Figure 4b. Figures a,b,c were generated using the program XFIT [55]. Structure 2000 8, 775-788DOI: (10.1016/S0969-2126(00)00164-7)

Figure 6 A modeling study for possible interaction between the 3-isoadeninyl group and the adenine-ring-binding site of diol dehydratase. (a) Stereoview superimposition of the structure of 3-isoadeninyl group on that of the enzyme-bound adeninylpentylcobalamin. Carbon atoms of the enzyme-bound adeninylpentylcobalamin are colored in pale violet-red. The position of the 3-isoadeninyl group and possible hydrogen bonds are shown as a green stick model. This figure was generated using the program MOLSCRIPT [57] and the rendering program RASTER3D [58]. (b) Hydrogen-bonding scheme between the 3-isoadeninyl group and the enzyme. Structure 2000 8, 775-788DOI: (10.1016/S0969-2126(00)00164-7)

Figure 7 A modeling study of superimposing the structure of adenosylcobalamin on that of the enzyme-bound adeninylpentylcobalamin. The adenosyl group of the coenzyme is shown as a green stick model. (a) Superimposed at the cobalamin moiety without cleavage of the Co–C bond. (b) Superimposed at both the cobalamin moiety and the adenine ring with the Co–C bond cleaved and keeping a Co–C distance minimum. (c) Superimposed as described in (b), but the ribose moiety of the adenosyl group is rotated around the glycosidic linkage so that C(5′) could come closest to C(1) of the substrate. This figure was generated using the program MOLSCRIPT [57] and the rendering program RASTER3D [58]. Structure 2000 8, 775-788DOI: (10.1016/S0969-2126(00)00164-7)

Figure 8 The proposed mechanism of action of diol dehydratase. The reaction with (S)-1,2-propanediol is illustrated. -Co-, cobalamin; Ade, 9-adeninyl group; Im, imidazole group of Hisα143. Structure 2000 8, 775-788DOI: (10.1016/S0969-2126(00)00164-7)